16 research outputs found

    Effect of the binding of the natural substrate 1-octen-3-ol on the OBP stability under pressure.

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    <p>Spectral transition curves were obtained by plotting the fluorescence center of spectral mass (panel A) and the maximum intensity of protein fluorescence emission (panel B) as a function of pressure. OBP proteins (dimer: circle, monomer: triangle) in 1.5 M GdnHCl, 20 mM Tris buffer pH 7.4 were incubated in the absence (•,▾) or in the presence (○,▿) of 1-octen-3-ol for 1 hour at 25°C before application of pressure. The best fit was made according to a two-step transition and the corresponding values are reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050489#pone-0050489-t001" target="_blank">Table 1</a>.</p

    Intrinsic Fluorescence of OBP.

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    <p>Intrinsic fluorescence emission spectra of native dimer OBP in the absence (dashed trace) and in the presence of 1.5 M GdnHCl (solid line) at 0.1 MPa.</p

    Protein fluorescence characterization in the presence of ANS.

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    <p>Panel A: ANS fluorescence emission spectra of dimer (full line) and monomer (dashed line) bOBP in the absence of substrate at 0.1 MPa. In the absence of proteins, the ANS probe exhibits only low fluorescence (dotted line). Panel B: ANS fluorescence intensity at 450 nm of the dimer OBP protein as a function of pressure in the absence (filled symbols) or in the presence (empty symbols) of 1 mM 1-octen-3-ol, respectively.</p

    Analysis of the distances between the center of mass of the two subunits during the simulation time.

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    <p>Variation of the distances of the center of mass of the two subunits of bOBP during the time, for simulations at 0.1 MPa (black), 250 MPa (blue) and 600 MPa (orange).</p

    Successive pressurization cycles of OBP binding protein in the presence of substrate.

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    <p>A) The protein fluorescence intensity at 338 nm is shown for increasing (filled symbols) and decreasing (empty symbols) pressures in a first (circles) and a second (squares) pressurization cycle. B) Pressure-jump induced relaxation kinetics. The p-jumps were from 185 to 350 MPa. The upper and lower traces reflect the kinetics in the first (close circles) and second pressurization cycle (open circle), respectively. C) Pressure-dependence of <i>k</i><sub>obs</sub> in the first and second pressurization cycles. The rate constants were determined by fitting the kinetic traces of 40 MPa downward pressure jumps to mono-exponential decays for the first (open circle) and the second (open square) cycles. D) Pressure dependence of the individual rate constants k<sub>f</sub> (filled symbols) and k<sub>u</sub> (empty symbols) of relaxation kinetics induced by downward pressure-jumps in the first (circles) and second (diamonds) pressurization cycles.</p

    Structural stability of dimer OBP after pressure treatment analyzed by size exclusion chromatography.

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    <p>Chromatograms of the dimer protein OBP in the absence of substrate before (solid line) and after (dashed line) pressure treatment up to 600 MPa and 250 MPa. The dash-dot-dot line represents the relative contribution of the monomer state of OBP assuming a Gaussian profile of elution for each oligomeric state of the protein. (<i>Inset</i>) The quantification of the monomer species was evaluated using bovine serum albumin (67 kDa), ovalbumin (36.9 kDa), chymotrypsinogen (23 kDa) and ribonuclease A (13.7 kDa) as standard proteins (filled circles). From the calculated partition coefficient K<sub>AV</sub>, molecular masses of 34.8 kDa and 18.97 kDa were determined for the initial dimer form (open square) and the pressure-induced dissociated form (open diamond), respectively.</p

    Zebrafish Prion Protein PrP2 Controls Collective Migration Process during Lateral Line Sensory System Development

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    <div><p>Prion protein is involved in severe neurodegenerative disorders but its physiological role is still in debate due to an absence of major developmental defects in knockout mice. Previous reports in zebrafish indicate that the two prion genes, <i>PrP1</i> and <i>PrP2</i>, are both involved in several steps of embryonic development thus providing a unique route to discover prion protein function. Here we investigate the role of PrP2 during development of a mechano-sensory system, the posterior lateral line, using morpholino knockdown and PrP2 targeted inactivation. We confirm the efficiency of the translation blocking morpholino at the protein level. Development of the posterior lateral line is altered in <i>PrP2</i> morphants, including nerve axonal outgrowth and primordium migration defects. Reduced neuromast deposition was observed in <i>PrP2</i> morphants as well as in <i>PrP2<sup>−/−</sup></i> mutants. Rosette formation defects were observed in <i>PrP2</i> morphants, strongly suggesting an abnormal primordium organization and reflecting loss of cell cohesion during migration of the primordium. In addition, the adherens junction proteins, E-cadherin and ß-catenin, were mis-localized after reduction of PrP2 expression and thus contribute to the primordium disorganization. Consequently, hair cell differentiation and number were affected and this resulted in reduced functional neuromasts. At later developmental stages, myelination of the posterior lateral line nerve was altered. Altogether, our study reports an essential role of PrP2 in collective migration process of the primordium and in neuromast formation, further implicating a role for prion protein in cell adhesion.</p></div

    Primodium disorganisation and absence of rosette formation.

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    <p><b>A′.</b> Schematic representation of a normal <i>claudinB-GFP</i> embryo at 30 hpf and detailed organization of the primordium with rosette structure. Red spot indicates a normal concentration point of actin. <b>A, B.</b> Phalloidin staining (Phalloidin-TRITC) in control embryo <i>claudinB-GFP</i>, at 30 hpf, is observed in muscle cells and within the primodium at the center of the rosette (arrows), on the apical side. <b>C, D.</b> Phalloidin-TRITC staining in <i>PrP2</i>-MO, no rosette structure is observed and no actin concentration is found. <b>E, F.</b> Higher magnification shows the co-localization of central actin concentration with <i>claudinB-GFP</i> at the rosette center in control. In morphants, cell disorganization is observed and no actin concentration is observed associated with the absence of a rosette. <b>G–I.</b> Phalloidin staining and DAPI nuclei labeling highlight the primordium and rosette center (arrows) in control embryos. <b>J–L.</b> In <i>PrP2<sup>−/−</sup></i> mutants, actin apical localization in rosette was severely reduced or barely detectable (arrow) and primordium organization at the periphery was impaired: loose cells were visible on the border (arrowheads). <b>I′, L′.</b> In <i>PrP2<sup>−/−</sup></i> mutant, the primordium position was often delayed and the first neuromast deposited close to the ear. <b>M</b>. Quantification of rosette number was established in control (n = 20), <i>PrP2</i>-MO (n = 84) and <i>PrP2<sup>−/−</sup></i> mutant (n = 28) using actin staining at the center, **: p<0.01, ***: p<0.001, Student t test. See also associated <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113331#pone.0113331.s002" target="_blank">Movies S1</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113331#pone.0113331.s006" target="_blank">S5</a>.</p
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